1. Field of the Invention
The present invention relates generally to the molecular biology of osteoclastogenesis. More specifically, the present invention relates to inhibitors of osteoclastogenesis and uses thereof.
2. Description of the Related Art
Nuclear Factor-kB (NF-kB) represents a group of five proteins, namely c-Rel, Rel A (p65), Rel B, NF-kB1 (p50 and p105), and NF-kB2 (p52). NF-kB is regulated by a family of inhibitors called IkB. In an inactive state, NF-kB is present in the cytoplasm as a heterotrimer consisting of p50, p65, and IkBa subunits. In response to an activation signal, the IkBa subunit is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded through the proteosomal pathway, thus exposing the nuclear localization signals on the p50-p65 heterodimer. The p65 is then phosphorylated, leading to nuclear translocation and binding to specific DNA sequence, which in turns results in gene transcription.
The p65 subunit of NF-kB, which contains at least two strong transactivation domains (TAD) within the C terminus (TA1 30 amino acid; TA2 90 amino acid), has been shown to undergo phosphorylation upon activation. The sites of phosphorylation and the kinase responsible for p65 phosphorylation remain controversial. For instance, phosphorylation at Ser 276 by protein kinase A, at Ser 529 by casein kinase II, at Ser 536 by IKK-b, and at serine 471 by PKC-e have been demonstrated. In addition, phosphorylation of p65-TAD by glycogen synthase kinase-3b and by Ca2+/calmodulin-dependent protein kinase IV have been demonstrated.
NF-kB has been shown to regulate the expression of a number of genes whose products are involved in inflammation, viral replication, carcinogenesis, anti-apoptosis, invasion and metastasis. These include anti-apoptosis genes, adhesion molecules, chemokines, inflammatory cytokines, and cell cycle regulatory genes. Thus agents that can suppress NF-kB activation have the potential to treat a variety of diseases that involves inflammation, apoptosis and carcinogenesis (Garg and Aggarwal, 2002; Karin and Lin, 2002; Zingarelli et al., 2003; Rosak et al., 2002).
Osteoclasts are multinucleated cells formed by the fusion of mononuclear progenitors of the monocyte/macrophage family and are the major resorptive cell of bone (Teitelbaum, 2000). In vitro maturation of macrophages into osteoclasts requires the presence of stromal cells or their osteoblast progeny (Udagawa et al., 1990). Extensive research in the last few years has indicated that these accessory cells express macrophage colony stimulating factor (MCSF) and receptor for activation of nuclear factor kappa B (NF-κB) (RANK) ligand (RANKL) and these are essential and sufficient to promote osteoclastogenesis. Besides macrophage colony stimulating factor and RANKL, several other inflammatory cytokines including TNF and IL-1β have been implicated in osteoclastogenesis, most likely through the osteoblastic modulation of RANKL and mCSF, respectively. The effects of parathyroid hormone and lipopolysaccharides on osteoclastogenesis are also mediated through expression of RANKL.
RANKL is a member of the TNF superfamily (Darnay & Aggarwal, 1999) that interacts with the cells surface receptor RANK, which in turn recruits TNF receptor-associated factors (TRAF)-1, 2, 3, 5 and 6 (Darnay et al., 1998; Wong et al., 1998). By receptor deletion analysis, sequential recruitment of TRAF6 and NF-κB-inducing kinase (NIK) by RANK was shown to lead to NF-κB activation, and recruitment of TRAF2 leads to JNK activation (Darnay et al., 1999; Lee et al., 1997).
That RANK can mediate osteoclastogenesis was first demonstrated by Hsu et al (Hsu et al., 1999). Further gene-deletion analysis of RANK, RANKL, and TRAF6 showed that these genes are positive regulators of osteoclastogenesis (Kong et al., 1999; Li et al., 2000; Lomaga et al., 1999), whereas osteoprotegerin (OPG), a decoy receptor for RANKL, was found to be a negative regulator of this process (Bucay et al., 1998; Lacey et al., 1998). Gene-deletion analysis also suggested a critical role of macrophage colony stimulating factor, c-fms (macrophage colony stimulating factor receptor) and Src in osteoclastogenesis (Dai et al., 2002; Tiffee et al., 1999; Xing et al., 2001).
Although RANKL is known to activate NF-κB, JNK, p42/p44 MAPK, and p38 MAPK (Darnay et al., 1999; Lee et al., 1997; Matsumoto et al., 2000; Zhang et al., 2001), how this cytokine mediates osteoclastogenesis is not fully understood. Furthermore agents that can suppress RANKL signaling can suppress osteoclastogenesis-induced bone loss. Because curcumin has been shown to suppress NF-κB activation induced by various inflammatory stimuli (Kumar et al., 1998, Bharti, 2003 #4; Singh & Aggarwal, 1995), inhibit the activation of IKK needed for NF-κB activation (Jobin et al., 1999; Pan et al., 2000; Plummer et al., 1999), and, found to be safe in humans even at 8 g per day (Cheng et al., 2001), the effect of curcumin on RANKL-induced NF-κB activation and on osteoclastogenesis in osteoclast precursor cells was examined.
The prior art is deficient in the demonstration that RANKL induces NF-κB activation through activation of IκB kinase (IKK), and IκBα phosphorylation and degradation and curcumin inhibits RANKL-induced NF-κB activation and osteoclastogenesis. The present invention fulfills this long-standing need and desire in the art.